This invention relates generally to a polymer membrane type ion-selective electrode, and more particularly, to a polymer membrane type ion-selective electrode suitable for monitoring polycationic macromolecules such as protamine.
Polymer membrane type ion-selective electrodes are now routinely used in commercial biomedical instruments to measure accurately levels of clinical important small ions, such as Ca.sup.++, Na.sup.+, K.sup.+, Li.sup.+, H.sup.+, and Cl, in undiluted serum, plasma, and whole blood. These ion-selective electrodes typically comprise a highly plasticized polymeric matrix material with an ion-exchange material or ion-complexing agent therein. The ion-exchange material may be a quaternary ammonium salt, such as tridodecyl methyl ammonium chloride (TDMAC).
Polyvinyl chloride (PVC) is a common polymeric membrane matrix material used in the art of solid-state or liquid-membrane electrodes for the detection of small ions (see, for example, U.S. Pat. No. 4,861,455 or Hartman, et al., "Chloride-Selective Liquid-Membrane Electrodes Based on Lipophilic Methyl-Tri-N-Alkyl-Ammonium Compounds and Their Applicability to Blood Serum Measurements," Mikrochimica Acta [Wein], 1978 II 235-246).
Efforts to develop similar sensors, including immuno-based biosensors, for the detection of large biomolecules, such as proteins or drugs, have thus far been unsuccessful. One of the most difficult problems has been identifying appropriate complexing agents and membrane chemistries that yield significant, specific and reversible electrochemical responses to the desired analyte. Even if a specific complexing agent is identified for a macromolecular biomolecule, whether the interaction with the macromolecular ion is strong enough to overcome the rather low mobility of a large ion to yield to significant electrochemical response remains in question. In theory, the sensitivity and selectivity of an ion-selective electrode membranes is governed by both the mobility of the analyte ion and the strength of the interaction between the ion-complexing agent and the analyte ion. In addition, strong interference resulting from a high concentration of small ions, such as chloride ions in a blood sample, may dictate the membrane's response.
U.S. Pat. No. 5,236,570, and its related patent application(s), disclose specially formulated polymer membranes which exhibit a large and reproducible potentionmetic response toward the polyanionic macromolecule heparin at clinically relevant concentrations. In preferred embodiments, heparin-selective membranes comprise poly(vinyl chloride) doped with a quaternary ammonium salt, such as tridodecyl methyl ammonium chloride, as an anion-complexing agent. The polyion response has been attributed to the development of a non-equilibrium steady-state-phase boundary potential change at the membrane/sample interface. The steady-state condition occurs when the flux of heparin extraction into the membrane from the sample solution via an ion-exchange reaction is equal to the flux of heparin into the bulk of the membrane. Because of the non-equilibrium response mechanism, the use of membranes with low plasticizer content (preferably less than 33%) yields sensors that respond to heparin at lower concentrations than conventional, more highly plasticized membranes (i.e., &gt;66%). In other embodiments, successful membranes comprising silicone rubber do not require a plasticizer to achieve the appropriate flux conditions.
Heparin, of course, is an analyte of particular clinical significance since heparin is the anticoagulant drug used almost universally in surgical procedures and extracorporeal therapies, and for the prevention of thromboembolism following surgery or childbirth. Currently, protamine sulfate is the only available compound used to reverse heparin-induced anticoagulation. Protamine sulfate is a polycationic peptide (average M.sub.r is 4,500) derived from salmon sperm, sometimes designated salmine protamine or n-protamine. The major constituent of protamine is the basic amino acid arginine, a highly alkaline cationic substance. The basic guanadinium groups of protamine complex electrostatically with the sulfonate groups of heparin to render the anticoagulant activity of the heparin ineffective. Unfortunately, the use of protamine frequently results in adverse hemodynamic and hematologic side effects such as hypotension, bradycardia, pulmonary artery hypertension, depressed oxygen consumption, thrombocytopenia with pulmonary platelet sequestration, and leukopenia. There is, therefore, a need in the art for a method of clinically detecting the concentration of protamine in a biological fluid, such as blood or plasma.
Protamine is also used as a titrant in commercially available blood-clotting instrumentation to determine accurately heparin levels in blood. Because there are no aromatic amino acid residues (e.g., tyrosine) in protamine, conventional UV absorption methods can not be used to detect protamine in solution. Protamine interacts with conventional protein reagents, such as the Folin-phenol reagent and Coomassie brilliant blue G-250, and therefore, can be measured via either the Lowry or Bradford methods [see, for example, Lowry, et al., J. Biol. Chem., Vol. 193, p. 265 (1951) or Bradford, et al., Anal. Biochem., Vol. 72, p. 248 (1976)]. However, neither of these methods are specific for protamine. In addition, protamine is devoid of catalytic and enzymatic activities which could form the basis of an assay. Consequently, there is a need in the art for a means of quantifying protamine in solution, particularly in a complex medium, such as blood fluids, wherein a variety of proteins and other ionic species may be present.
Negatively charged lipophilic anions, such as tetraphenylborate derivatives, have been used in the art to make polymer membrane-type electrodes which are sensitive to organic cations including organic drug species, vitamins, and amino acids. These known sensors typically respond to hydrophobic organic cations based on their relative solubility in the organic membrane phase. There is, however, a need for a sensor which can detect a very hydrophilic species, such as protamine, which would not be expected to partition into the organic phase of a polymer membrane-type sensor.
It is, therefore, an object of this invention to provide an electrochemical sensor for ionic macromolecules.
It is another object of this invention to provide an electrochemical sensor for direct measurement of ionic macromolecules in whole blood or plasma.
It is also an object of this invention to provide an electrochemical sensor for direct measurement of ionic macromolecules in whole blood or plasma which is accurate over the expected clinically relevant concentration range.
It is a further object of this invention to provide an electrochemical sensor for direct measurement of ionic macromolecules in whole blood or plasma which possesses adequate dynamic response characteristics, i.e., responds rapidly to a change in ion concentration and returns promptly to baseline, so that it is suitable for continuous in vivo monitoring.
It is additionally an object of this invention to provide a polymeric membrane electrode having specific selectivity for biologically important macromolecules even in the presence of Cl.sup.- and other anionic impurities.